Bottom Line:
Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease.By contrast, embryonic epithelia show normal junctions.Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

ABSTRACTPlakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

fig8: Triton X-100 solubility of junctional proteins in the hearts of wt and plakophilin 2 mutant (pkp2−/−) embryos at E10.75. Triton X-100 insoluble and soluble proteins were subjected to SDS-PAGE and the relative amounts of specific proteins were demonstrated by Western blotting using antibodies to junctional proteins as listed. Note that a large proportion of desmoplakin and desmoglein 2 and, to a lesser degree, plakoglobin, were not associated with the Triton X-100 insoluble (i.e., cytoskeleton-associated) fraction in the plakophilin 2–deficient hearts. Plakophilin 2 was absent in mutant hearts, and β-catenin as well as N-cadherin did not show marked differences between wt and pkp2−/− animals.

Mentions:
We generated a mutation of the plakophilin 2 gene by homologous recombination in embryonic stem (ES) cells. In the targeting vector, a neo cassette inserted in opposite transcriptional orientation replaced a 7.8-kb genomic fragment from the NotI site in exon 1 to the BamHI site in intron 1 (Fig. 1 a). Homologous recombination events were identified by Southern blot analyses (Fig. 1 b): an external probe (ext) yielded a novel Xba fragment of 10 kb in the mutant, Δpkp2 (the wild-type [wt] fragment is 16 kb), and the neo probe produced a 4.5-kb HindIII fragment. We generated a mutation in the plakophilin 2 gene after homologous recombination because insertion of neo prevents splicing between exon 1 and 2 and leads to an early stop of plakophilin 2 translation after 43 aa. Using two lines of mutant ES cells, we produced plakophilin 2 mutant chimeric and heterozygous mice that were healthy and fertile. However, matings between heterozygous mice produced no live plakophilin 2–deficient offspring, implying that the mutant embryos died during embryogenesis. To determine the time of death, embryos from different developmental stages were genotyped by PCR (Fig. 1 c) and inspected visually. Up to day 10.75 of embryogenesis (E10.75), the expected Mendelian ratio of homozygous mutant embryos was observed (Table I); however the mutant embryos showed blood accumulation in the pericardial and peritoneal cavities. At E11.5, the number of viable plakophilin 2 −/− embryos declined, as judged by PCR genotyping and lack of heart beating. Western blot analyses using an antibody against the COOH terminus of plakophilin 2 indicated absence of full-length or truncated protein in E10.75 plakophilin 2 −/− embryos (Fig. 1 d). In wt embryos at E10.75, plakophilin 2 was prominent in cardiomyocytes of the atrium and the ventricle of the heart (Fig. 1 e), and expression was lost in the mutation embryos (see also below, Fig. 4 Bd′, and Fig. 8). At E13.75, plakophilin 2 was intensely synthesized in the wt cardiomyocytes, but less in the surrounding epicard (Fig. 1 f).

fig8: Triton X-100 solubility of junctional proteins in the hearts of wt and plakophilin 2 mutant (pkp2−/−) embryos at E10.75. Triton X-100 insoluble and soluble proteins were subjected to SDS-PAGE and the relative amounts of specific proteins were demonstrated by Western blotting using antibodies to junctional proteins as listed. Note that a large proportion of desmoplakin and desmoglein 2 and, to a lesser degree, plakoglobin, were not associated with the Triton X-100 insoluble (i.e., cytoskeleton-associated) fraction in the plakophilin 2–deficient hearts. Plakophilin 2 was absent in mutant hearts, and β-catenin as well as N-cadherin did not show marked differences between wt and pkp2−/− animals.

Mentions:
We generated a mutation of the plakophilin 2 gene by homologous recombination in embryonic stem (ES) cells. In the targeting vector, a neo cassette inserted in opposite transcriptional orientation replaced a 7.8-kb genomic fragment from the NotI site in exon 1 to the BamHI site in intron 1 (Fig. 1 a). Homologous recombination events were identified by Southern blot analyses (Fig. 1 b): an external probe (ext) yielded a novel Xba fragment of 10 kb in the mutant, Δpkp2 (the wild-type [wt] fragment is 16 kb), and the neo probe produced a 4.5-kb HindIII fragment. We generated a mutation in the plakophilin 2 gene after homologous recombination because insertion of neo prevents splicing between exon 1 and 2 and leads to an early stop of plakophilin 2 translation after 43 aa. Using two lines of mutant ES cells, we produced plakophilin 2 mutant chimeric and heterozygous mice that were healthy and fertile. However, matings between heterozygous mice produced no live plakophilin 2–deficient offspring, implying that the mutant embryos died during embryogenesis. To determine the time of death, embryos from different developmental stages were genotyped by PCR (Fig. 1 c) and inspected visually. Up to day 10.75 of embryogenesis (E10.75), the expected Mendelian ratio of homozygous mutant embryos was observed (Table I); however the mutant embryos showed blood accumulation in the pericardial and peritoneal cavities. At E11.5, the number of viable plakophilin 2 −/− embryos declined, as judged by PCR genotyping and lack of heart beating. Western blot analyses using an antibody against the COOH terminus of plakophilin 2 indicated absence of full-length or truncated protein in E10.75 plakophilin 2 −/− embryos (Fig. 1 d). In wt embryos at E10.75, plakophilin 2 was prominent in cardiomyocytes of the atrium and the ventricle of the heart (Fig. 1 e), and expression was lost in the mutation embryos (see also below, Fig. 4 Bd′, and Fig. 8). At E13.75, plakophilin 2 was intensely synthesized in the wt cardiomyocytes, but less in the surrounding epicard (Fig. 1 f).

Bottom Line:
Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease.By contrast, embryonic epithelia show normal junctions.Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

ABSTRACTPlakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.